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Contents
part one
General Embryology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
chapter 1
Gametogenesis: Conversion of Germ Cells Into Male and
Female Gametes ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
chapter 2
First Week of Development: Ovulation to Implantation ............ . . . . . . .
31
chapter 3
Second Week of Development: Bilaminar Germ Disc ............ . . . . . . . . . .
51
chapter 4
Third Week of Development: Trilaminar Germ Disc ............. . . . . . . . . . .
65
chapter 5
Third to Eighth Week: The Embryonic Period ............... . . . . . . . . . . . . . . . .
87
chapter 6
Third Month to Birth: The Fetus and Placenta ................... . . . . . . . . . . . .
117
chapter 7
Birth Defects and Prenatal Diagnosis ................... . . . . . . . . . . . . . . . . . . . . .
149
part two
Special Embryology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
169
chapter 8
Skeletal System ............................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
171
ix
x
Contents
chapter 9
Muscular System ........................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
199
chapter 10
Body Cavities ............................ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
211
chapter 11
Cardiovascular System ......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
223
chapter 12
Respiratory System ........................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
275
chapter 13
Digestive System ........................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
285
chapter 14
Urogenital System .......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
321
chapter 15
Head and Neck ; ............................ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
363
chapter 16
Ear ................................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
403
chapter 17
Eye ................................ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
415
chapter 18
Integumentary System .......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
427
chapter 19
Central Nervous System ......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
433
part three
Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
483
Answers to Problems .......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
485
Figure Credits .......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
499
Index ................................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
507
Preface
The ninth edition of Langman’s Medical Embryology adheres to the tradition
established by the original publication—it provides a concise but thorough description of embryology and its clinical signiﬁcance, an awareness of which is
essential in the diagnosis and prevention of birth defects. Recent advances in genetics, developmental biology, maternal-fetal medicine, and public health have
signiﬁcantly increased our knowledge of embryology and its relevance. Because
birth defects are the leading cause of infant mortality and a major contributor to
disabilities, and because new prevention strategies have been developed, understanding the principles of embryology is important for health care professionals.
To accomplish its goal, Langman’s Medical Embryology retains its unique approach of combining an economy of text with excellent diagrams and scanning
electron micrographs. It reinforces basic embryologic concepts by providing
numerous clinical examples that result from abnormalities in developmental
processes. The following pedagogic features and updates in the ninth edition
help facilitate student learning:
Organization of Material: Langman’s Medical Embryology is organized into two
parts. The ﬁrst provides an overview of early development from gametogenesis
through the embryonic period; also included in this section are chapters on
placental and fetal development and prenatal diagnosis and birth defects. The
second part of the text provides a description of the fundamental processes of
embryogenesis for each organ system.
Molecular Biology: New information is provided about the molecular basis of
normal and abnormal development.
Extensive Art Program: This edition features almost 400 illustrations, including new 4-color line drawings, scanning electron micrographs, and ultrasound
images.
Clinical Correlates: In addition to describing normal events, each chapter contains clinical correlates that appear in highlighted boxes. This material is designed to provide information about birth defects and other clinical entities that
are directly related to embryologic concepts.
vii
viii
Preface
Summary: At the end of each chapter is a summary that serves as a concise
review of the key points described in detail throughout the chapter.
Problems to Solve: These problems test a student’s ability to apply the information covered in a particular chapter. Detailed answers are provided in an
appendix in the back of the book.
Simbryo: New to this edition, Simbryo, located in the back of the book, is
an interactive CD-ROM that demonstrates normal embryologic events and the
origins of some birth defects. This unique educational tool offers six original
vector art animation modules to illustrate the complex, three-dimensional aspects of embryology. Modules include normal early development as well as
head and neck, cardiovascular, gastrointestinal, genitourinary, and pulmonary
system development.
Connection Web Site: This student and instructor site (http://connection.
LWW.com/go/sadler) provides updates on new advances in the ﬁeld and a syllabus designed for use with the book. The syllabus contains objectives and
deﬁnitions of key terms organized by chapters and the “bottom line,” which
provides a synopsis of the most basic information that students should have
mastered from their studies.
I hope you ﬁnd this edition of Langman’s Medical Embryology to be an
excellent resource. Together, the textbook, CD, and connection site provide a
user-friendly and innovative approach to learning embryology and its clinical
relevance.
T. W. Sadler
Twin Bridges, Montana
p a r t
o n e
General
Embryology
1
c h a p t e r
1
Gametogenesis: Conversion
of Germ Cells Into Male and
Female Gametes
Primordial Germ Cells
Development begins with fertilization, the process by which the male gamete, the sperm, and the
female gamete, the oocyte, unite to give rise to a zygote.
Gametes are derived from primordial germ cells (PGCs)
that are formed in the epiblast during the second week
and that move to the wall of the yolk sac (Fig. 1.1). During
the fourth week these cells begin to migrate from the yolk
sac toward the developing gonads, where they arrive by the
end of the ﬁfth week. Mitotic divisions increase their number
during their migration and also when they arrive in the gonad.
In preparation for fertilization, germ cells undergo gametogenesis,
which includes meiosis, to reduce the number of chromosomes and
cytodifferentiation to complete their maturation.
CLINICAL CORRELATE
Primordial Germ Cells (PGCs) and Teratomas
Teratomas are tumors of disputed origin that often contain a variety
of tissues, such as bone, hair, muscle, gut epithelia, and others. It is
thought that these tumors arise from a pluripotent stem cell that can
differentiate into any of the three germ layers or their derivatives.
3
4
Part One: General Embryology
Figure 1.1 An embryo at the end of the third week, showing the position of primordial
germ cells in the wall of the yolk sac, close to the attachment of the future umbilical
cord. From this location, these cells migrate to the developing gonad.
Some evidence suggests that PGCs that have strayed from their normal migratory paths could be responsible for some of these tumors. Another source
is epiblast cells migrating through the primitive streak during gastrulation
(see page 80).
The Chromosome Theory of Inheritance
Traits of a new individual are determined by speciﬁc genes on chromosomes
inherited from the father and the mother. Humans have approximately 35,000
genes on 46 chromosomes. Genes on the same chromosome tend to be inherited together and so are known as linked genes. In somatic cells, chromosomes
appear as 23 homologous pairs to form the diploid number of 46. There are
22 pairs of matching chromosomes, the autosomes, and one pair of sex chromosomes. If the sex pair is XX, the individual is genetically female; if the pair is
XY, the individual is genetically male. One chromosome of each pair is derived
from the maternal gamete, the oocyte, and one from the paternal gamete, the
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
5
sperm. Thus each gamete contains a haploid number of 23 chromosomes, and
the union of the gametes at fertilization restores the diploid number of 46.
MITOSIS
Mitosis is the process whereby one cell divides, giving rise to two daughter
cells that are genetically identical to the parent cell (Fig. 1.2). Each daughter
cell receives the complete complement of 46 chromosomes. Before a cell enters
mitosis, each chromosome replicates its deoxyribonucleic acid (DNA). During
this replication phase the chromosomes are extremely long, they are spread
diffusely through the nucleus, and they cannot be recognized with the light microscope. With the onset of mitosis the chromosomes begin to coil, contract,
and condense; these events mark the beginning of prophase. Each chromosome now consists of two parallel subunits, chromatids, that are joined at a
narrow region common to both called the centromere. Throughout prophase
the chromosomes continue to condense, shorten, and thicken (Fig. 1.2A),
but only at prometaphase do the chromatids become distinguishable
(Fig. 1.2B). During metaphase the chromosomes line up in the equatorial plane,
Figure 1.2 Various stages of mitosis. In prophase, chromosomes are visible as slender threads. Doubled chromatids become clearly visible as individual units during
metaphase. At no time during division do members of a chromosome pair unite. Blue,
paternal chromosomes; red, maternal chromosomes.
6
Part One: General Embryology
and their doubled structure is clearly visible (Fig. 1.2C ). Each is attached by
microtubules extending from the centromere to the centriole, forming the mitotic spindle. Soon the centromere of each chromosome divides, marking the
beginning of anaphase, followed by migration of chromatids to opposite poles
of the spindle. Finally, during telophase, chromosomes uncoil and lengthen,
the nuclear envelope reforms, and the cytoplasm divides (Fig. 1.2, D and E ).
Each daughter cell receives half of all doubled chromosome material and thus
maintains the same number of chromosomes as the mother cell.
MEIOSIS
Meiosis is the cell division that takes place in the germ cells to generate male
and female gametes, sperm and egg cells, respectively. Meiosis requires two cell
divisions, meiosis I and meiosis II, to reduce the number of chromosomes to
the haploid number of 23 (Fig. 1.3). As in mitosis, male and female germ cells
(spermatocytes and primary oocytes) at the beginning of meiosis I replicate
their DNA so that each of the 46 chromosomes is duplicated into sister chromatids. In contrast to mitosis, however, homologous chromosomes then align
themselves in pairs, a process called synapsis. The pairing is exact and point
for point except for the XY combination. Homologous pairs then separate into
two daughter cells. Shortly thereafter meiosis II separates sister chromatids.
Each gamete then contains 23 chromosomes.
Crossover
Crossovers, critical events in meiosis I, are the interchange of chromatid segments between paired homologous chromosomes (Fig. 1.3C ). Segments of
chromatids break and are exchanged as homologous chromosomes separate.
As separation occurs, points of interchange are temporarily united and form an
X-like structure, a chiasma (Fig. 1.3C ). The approximately 30 to 40 crossovers
(one or two per chromosome) with each meiotic I division are most frequent
between genes that are far apart on a chromosome.
As a result of meiotic divisions, (a) genetic variability is enhanced through
crossover, which redistributes genetic material, and through random distribution of homologous chromosomes to the daughter cells; and (b) each germ cell
contains a haploid number of chromosomes, so that at fertilization the diploid
number of 46 is restored.
Polar Bodies
Also during meiosis one primary oocyte gives rise to four daughter cells, each
with 22 plus 1 X chromosomes (Fig. 1.4A). However, only one of these develops
into a mature gamete, the oocyte; the other three, the polar bodies, receive
little cytoplasm and degenerate during subsequent development. Similarly, one
primary spermatocyte gives rise to four daughter cells, two with 22 plus 1
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
7
Figure 1.3 First and second meiotic divisions. A. Homologous chromosomes approach
each other. B. Homologous chromosomes pair, and each member of the pair consists of
two chromatids. C. Intimately paired homologous chromosomes interchange chromatid
fragments (crossover). Note the chiasma. D. Double-structured chromosomes pull apart.
E. Anaphase of the ﬁrst meiotic division. F and G. During the second meiotic division,
the double-structured chromosomes split at the centromere. At completion of division,
chromosomes in each of the four daughter cells are different from each other.
X chromosomes and two with 22 plus 1 Y chromosomes (Fig. 1.4B ). However,
in contrast to oocyte formation, all four develop into mature gametes.
CLINICAL CORRELATES
Birth Defects and Spontaneous Abortions:
Chromosomal and Genetic Factors
Chromosomal abnormalities, which may be numerical or structural, are
important causes of birth defects and spontaneous abortions. It is estimated
that 50% of conceptions end in spontaneous abortion and that 50% of these
8
Part One: General Embryology
Figure 1.4 Events occurring during the ﬁrst and second maturation divisions. A. The
primitive female germ cell (primary oocyte) produces only one mature gamete, the mature oocyte. B. The primitive male germ cell (primary spermatocyte) produces four spermatids, all of which develop into spermatozoa.
abortuses have major chromosomal abnormalities. Thus approximately 25%
of conceptuses have a major chromosomal defect. The most common chromosomal abnormalities in abortuses are 45,X (Turner syndrome), triploidy,
and trisomy 16. Chromosomal abnormalities account for 7% of major birth
defects, and gene mutations account for an additional 8%.
Numerical Abnormalities
The normal human somatic cell contains 46 chromosomes; the normal gamete contains 23. Normal somatic cells are diploid, or 2n; normal gametes
are haploid, or n. Euploid refers to any exact multiple of n, e.g., diploid or
triploid. Aneuploid refers to any chromosome number that is not euploid; it is
usually applied when an extra chromosome is present (trisomy) or when one
is missing (monosomy). Abnormalities in chromosome number may originate during meiotic or mitotic divisions. In meiosis, two members of a pair
of homologous chromosomes normally separate during the ﬁrst meiotic division so that each daughter cell receives one member of each pair (Fig. 1.5A).
Sometimes, however, separation does not occur (nondisjunction), and both
members of a pair move into one cell (Fig. 1.5, B and C ). As a result of
nondisjunction of the chromosomes, one cell receives 24 chromosomes,
and the other receives 22 instead of the normal 23. When, at fertilization, a gamete having 23 chromosomes fuses with a gamete having 24 or
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
9
Figure 1.5 A. Normal maturation divisions. B. Nondisjunction in the ﬁrst meiotic division. C. Nondisjunction in the second meiotic division.
22 chromosomes, the result is an individual with either 47 chromosomes
(trisomy) or 45 chromosomes (monosomy). Nondisjunction, which occurs
during either the ﬁrst or the second meiotic division of the germ cells, may
involve the autosomes or sex chromosomes. In women, the incidence of
chromosomal abnormalities, including nondisjunction, increases with age,
especially at 35 years and older.
Occasionally nondisjunction occurs during mitosis (mitotic nondisjunction) in an embryonic cell during the earliest cell divisions. Such conditions
produce mosaicism, with some cells having an abnormal chromosome number and others being normal. Affected individuals may exhibit few or many
of the characteristics of a particular syndrome, depending on the number of
cells involved and their distribution.
Sometimes chromosomes break, and pieces of one chromosome attach
to another. Such translocations may be balanced, in which case breakage and
reunion occur between two chromosomes but no critical genetic material is
lost and individuals are normal; or they may be unbalanced, in which case
part of one chromosome is lost and an altered phenotype is produced. For
example, unbalanced translocations between the long arms of chromosomes
14 and 21 during meiosis I or II produce gametes with an extra copy of chromosome 21, one of the causes of Down syndrome (Fig. 1.6). Translocations
10
Part One: General Embryology
A
14
21
t(14;21)
Figure 1.6 A. Translocation of the long arms of chromosomes 14 and 21 at the centromere. Loss of the short arms is not clinically signiﬁcant, and these individuals are
clinically normal, although they are at risk for producing offspring with unbalanced
translocations. B. Karyotype of translocation of chromosome 21 onto 14, resulting in
Down syndrome.
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
11
Figure 1.7 Karyotype of trisomy 21 (arrow), Down syndrome.
are particularly common between chromosomes 13, 14, 15, 21, and 22 because they cluster during meiosis.
TRISOMY
21 (DOWN SYNDROME)
Down syndrome is usually caused by an extra copy of chromosome 21 (trisomy 21, Fig. 1.7). Features of children with Down syndrome include growth
retardation; varying degrees of mental retardation; craniofacial abnormalities,
including upward slanting eyes, epicanthal folds (extra skin folds at the medial
corners of the eyes), ﬂat facies, and small ears; cardiac defects; and hypotonia
(Fig. 1.8). These individuals also have relatively high incidences of leukemia,
infections, thyroid dysfunction, and premature aging. Furthermore, nearly
all develop signs of Alzheimer’s disease after age 35. In 95% of cases, the
syndrome is caused by trisomy 21 resulting from meiotic nondisjunction, and
in 75% of these instances, nondisjunction occurs during oocyte formation.
The incidence of Down syndrome is approximately 1 in 2000 conceptuses
for women under age 25. This risk increases with maternal age to 1 in 300 at
age 35 and 1 in 100 at age 40.
In approximately 4% of cases of Down syndrome, there is an unbalanced translocation between chromosome 21 and chromosome 13, 14, or 15
(Fig. 1.6). The ﬁnal 1% are caused by mosaicism resulting from mitotic
12
Part One: General Embryology
Figure 1.8 A and B. Children with Down syndrome, which is characterized by a ﬂat,
broad face, oblique palpebral ﬁssures, epicanthus, and furrowed lower lip. C. Another
characteristic of Down syndrome is a broad hand with single transverse or simian crease.
Many children with Down syndrome are mentally retarded and have congenital heart
abnormalities.
nondisjunction. These individuals have some cells with a normal chromosome number and some that are aneuploid. They may exhibit few or many
of the characteristics of Down syndrome.
TRISOMY
18
Patients with trisomy 18 show the following features: mental retardation, congenital heart defects, low-set ears, and ﬂexion of ﬁngers and hands (Fig. 1.9). In
addition, patients frequently show micrognathia, renal anomalies, syndactyly,
and malformations of the skeletal system. The incidence of this condition is
approximately 1 in 5000 newborns. Eighty-ﬁve percent are lost between 10
weeks of gestation and term, whereas those born alive usually die by age
2 months.
13
The main abnormalities of trisomy 13 are mental retardation, holoprosencephaly, congenital heart defects, deafness, cleft lip and palate,
and eye defects, such as microphthalmia, anophthalmia, and coloboma
(Fig. 1.10). The incidence of this abnormality is approximately 1 in 20,000
live births, and over 90% of the infants die in the ﬁrst month after
birth.
TRISOMY
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
13
Figure 1.9 Photograph of child with trisomy 18. Note the prominent occiput, cleft lip,
micrognathia, low-set ears, and one or more ﬂexed ﬁngers.
Figure 1.10 A. Child with trisomy 13. Note the cleft lip and palate, the sloping forehead,
and microphthalmia. B. The syndrome is commonly accompanied by polydactyly.
KLINEFELTER SYNDROME
The clinical features of Klinefelter syndrome, found only in males and usually
detected at puberty, are sterility, testicular atrophy, hyalinization of the seminiferous tubules, and usually gynecomastia. The cells have 47 chromosomes
with a sex chromosomal complement of the XXY type, and a sex chromatin
body (Barr body: formed by condensation of an inactivated sex chromosome; a Barr body is also present in normal females) is found in 80% of cases
(Fig. 1.11). The incidence is approximately 1 in 500 males. Nondisjunction of
the XX homologues is the most common causative event. Occasionally, patients with Klinefelter syndrome have 48 chromosomes: 44 autosomes and
four sex chromosomes (XXXY). Although mental retardation is not generally
14
Part One: General Embryology
Figure 1.11 Patient with Klinefelter syndrome showing normal phallus development
but gynecomastia (enlarged breasts).
part of the syndrome, the more X chromosomes there are, the more likely
there will be some degree of mental impairment.
TURNER SYNDROME
Turner syndrome, with a 45,X karyotype, is the only monosomy compatible with life. Even then, 98% of all fetuses with the syndrome are spontaneously aborted. The few that survive are unmistakably female in appearance
(Fig. 1.12) and are characterized by the absence of ovaries (gonadal dysgenesis) and short stature. Other common associated abnormalities are webbed
neck, lymphedema of the extremities, skeletal deformities, and a broad chest
with widely spaced nipples. Approximately 55% of affected women are monosomic for the X and chromatin body negative because of nondisjunction. In
80% of these women, nondisjunction in the male gamete is the cause. In
the remainder of women, structural abnormalities of the X chromosome or
mitotic nondisjunction resulting in mosaicism are the cause.
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
15
Figure 1.12 Patient with Turner syndrome. The main characteristics are webbed neck,
short stature, broad chest, and absence of sexual maturation.
TRIPLE X SYNDROME
Patients with triple X syndrome are infantite, with scanty menses and some
degree of mental retardation. They have two sex chromatin bodies in their
cells.
Structural Abnormalities
Structural chromosome abnormalities, which involve one or more chromosomes, usually result from chromosome breakage. Breaks are caused by
environmental factors, such as viruses, radiation, and drugs. The result of
breakage depends on what happens to the broken pieces. In some cases, the
broken piece of a chromosome is lost, and the infant with partial deletion of
a chromosome is abnormal. A well-known syndrome, caused by partial deletion of the short arm of chromosome 5, is the cri-du-chat syndrome. Such
children have a catlike cry, microcephaly, mental retardation, and congenital
heart disease. Many other relatively rare syndromes are known to result from
a partial chromosome loss.
Microdeletions, spanning only a few contiguous genes, may result in
microdeletion syndrome or contiguous gene syndrome. Sites where these
deletions occur, called contiguous gene complexes, can be identiﬁed by
high-resolution chromosome banding. An example of a microdeletion
16
Part One: General Embryology
Figure 1.13 Patient with Angelman syndrome resulting from a microdeletion on maternal chromosome 15. If the defect is inherited on the paternal chromosome, Prader-Willi
syndrome occurs (Fig. 1.14).
occurs on the long arm of chromosome 15 (15q11–15q13). Inheriting the
deletion on the maternal chromosome results in Angelman syndrome, and
the children are mentally retarded, cannot speak, exhibit poor motor development, and are prone to unprovoked and prolonged periods of laughter
(Fig. 1.13). If the defect is inherited on the paternal chromosome, Prader-Willi
syndrome is produced; affected individuals are characterized by hypotonia,
obesity, mental retardation, hypogonadism, and cryptorchidism (Fig. 1.14).
Characteristics that are differentially expressed depending upon whether the
genetic material is inherited from the mother or the father are examples of
genomic imprinting. Other contiguous gene syndromes may be inherited
from either parent, including Miller-Dieker syndrome (lissencephaly, developmental delay, seizures, and cardiac and facial abnormalities resulting from a
deletion at 17p13) and most cases of velocardiofacial (Shprintzen) syndrome
(palatal defects, conotruncal heart defects, speech delay, learning disorders,
and schizophrenia-like disorder resulting from a deletion in 22q11).
Fragile sites are regions of chromosomes that demonstrate a propensity
to separate or break under certain cell manipulations. For example, fragile
sites can be revealed by culturing lymphocytes in folate-deﬁcient medium.
Although numerous fragile sites have been deﬁned and consist of CGG repeats, only the site on the long arm of the X chromosome (Xq27) has been
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
17
Figure 1.14 Patient with Prader-Willi syndrome resulting from a microdeletion on paternal chromosome 15. If the defect is inherited on the maternal chromosome, Angelman
syndrome occurs (Fig. 1.13).
correlated with an altered phenotype and is called the fragile X syndrome.
Fragile X syndrome is characterized by mental retardation, large ears, prominent jaw, and pale blue irides. Males are affected more often than females
(1/1000 versus 1/2000), which may account for the preponderance of males
among the mentally retarded. Fragile X syndrome is second only to Down
syndrome as a cause of mental retardation because of chromosomal abnormalities.
Gene Mutations
Many congenital formations in humans are inherited, and some show a clear
mendelian pattern of inheritance. Many birth defects are directly attributable
to a change in the structure or function of a single gene, hence the name single
gene mutation. This type of defect is estimated to account for approximately
8% of all human malformations.
18
Part One: General Embryology
With the exception of the X and Y chromosomes in the male, genes exist
as pairs, or alleles, so that there are two doses for each genetic determinant,
one from the mother and one from the father. If a mutant gene produces an
abnormality in a single dose, despite the presence of a normal allele, it is a
dominant mutation. If both alleles must be abnormal (double dose) or if the
mutation is X-linked in the male, it is a recessive mutation. Gradations in the
effects of mutant genes may be a result of modifying factors.
The application of molecular biological techniques has increased our
knowledge of genes responsible for normal development. In turn, genetic
analysis of human syndromes has shown that mutations in many of these
same genes are responsible for some congenital abnormalities and childhood
diseases. Thus, the link between key genes in development and their role in
clinical syndromes is becoming clearer.
In addition to causing congenital malformations, mutations can result in
inborn errors of metabolism. These diseases, among which phenylketonuria,
homocystinuria, and galactosemia are the best known, are frequently accompanied by or cause various degrees of mental retardation.
Diagnostic Techniques for Identifying Genetic Abnormalities
Cytogenetic analysis is used to assess chromosome number and integrity.
The technique requires dividing cells, which usually means establishing cell
cultures that are arrested in metaphase by chemical treatment. Chromosomes
are stained with Giemsa stain to reveal light and dark banding patterns
(G-bands; Fig. 1.6) unique for each chromosome. Each band represents 5 to
10 × 106 base pairs of DNA, which may include a few to several hundred genes.
Recently, high resolution metaphase banding techniques have been developed that demonstrate greater numbers of bands representing even smaller
pieces of DNA, thereby facilitating diagnosis of small deletions.
New molecular techniques, such as ﬂuorescence in situ hybridization
(FISH), use speciﬁc DNA probes to identify ploidy for a few selected chromosomes. Fluorescent probes are hybridized to chromosomes or genetic
loci using cells on a slide, and the results are visualized with a ﬂuorescence
microscope (Fig.1.15). Spectral karyotype analysis is a technique in which
every chromosome is hybridized to a unique ﬂuorescent probe of a different
color. Results are then analyzed by a computer.
Morphological Changes During Maturation
of the Gametes
OOGENESIS
Maturation of Oocytes Begins Before Birth
Once primordial germ cells have arrived in the gonad of a genetic female, they
differentiate into oogonia (Fig. 1.16, A and B). These cells undergo a number
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
19
Figure 1.15 Fluorescence in situ hybridization (FISH) using a probe for chromosome
21. Two interphase cells and a metaphase spread of chromosomes are shown; each has
three domains, indicated by the probe, characteristic of trisomy 21 (Down syndrome).
Figure 1.16 Differentiation of primordial germ cells into oogonia begins shortly after
their arrival in the ovary. By the third month of development, some oogonia give rise
to primary oocytes that enter prophase of the ﬁrst meiotic division. This prophase may
last 40 or more years and ﬁnishes only when the cell begins its ﬁnal maturation. During
this period it carries 46 double-structured chromosomes.
20
Part One: General Embryology
Surface epithelium of ovary
Primary oocyte in
prophase
Flat
epithelial
cell
Resting primary oocyte
(diplotene stage)
Follicular cell
Oogonia
Primary
oocytes in
prophase
of 1st
meiotic
division
A
C
B
4th month
7th month
Newborn
Figure 1.17 Segment of the ovary at different stages of development. A. Oogonia are
grouped in clusters in the cortical part of the ovary. Some show mitosis; others have
differentiated into primary oocytes and entered prophase of the ﬁrst meiotic division. B.
Almost all oogonia are transformed into primary oocytes in prophase of the ﬁrst meiotic
division. C. There are no oogonia. Each primary oocyte is surrounded by a single layer
of follicular cells, forming the primordial follicle. Oocytes have entered the diplotene
stage of prophase, in which they remain until just before ovulation. Only then do they
enter metaphase of the ﬁrst meiotic division.
of mitotic divisions and, by the end of the third month, are arranged in clusters
surrounded by a layer of ﬂat epithelial cells (Fig. 1.17 and 1.18). Whereas all
of the oogonia in one cluster are probably derived from a single cell, the ﬂat
epithelial cells, known as follicular cells, originate from surface epithelium
covering the ovary.
The majority of oogonia continue to divide by mitosis, but some of them
arrest their cell division in prophase of meiosis I and form primary oocytes
(Figs. 1.16C and 1.17A). During the next few months, oogonia increase rapidly
in number, and by the ﬁfth month of prenatal development, the total number
of germ cells in the ovary reaches its maximum, estimated at 7 million. At this
time, cell death begins, and many oogonia as well as primary oocytes become
atretic. By the seventh month, the majority of oogonia have degenerated except
for a few near the surface. All surviving primary oocytes have entered prophase
of meiosis I, and most of them are individually surrounded by a layer of ﬂat
epithelial cells (Fig. 1.17B). A primary oocyte, together with its surrounding ﬂat
epithelial cells, is known as a primordial follicle (Fig. 1.19A).
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
21
Figure 1.18 A. Primordial follicle consisting of a primary oocyte surrounded by a layer
of ﬂattened epithelial cells. B. Early primary or preantral stage follicle recruited from
the pool of primordial follicles. As the follicle grows, follicular cells become cuboidal
and begin to secrete the zona pellucida, which is visible in irregular patches on the
surface of the oocyte. C. Mature primary (preantral) follicle with follicular cells forming
a stratiﬁed layer of granulosa cells around the oocyte and the presence of a well-deﬁned
zona pellucida.
Maturation of Oocytes Continues at Puberty
Near the time of birth, all primary oocytes have started prophase of meiosis I,
but instead of proceeding into metaphase, they enter the diplotene stage, a
resting stage during prophase that is characterized by a lacy network of chromatin (Fig. 1.17C ). Primary oocytes remain in prophase and do not ﬁnish
their ﬁrst meiotic division before puberty is reached, apparently because of
oocyte maturation inhibitor (OMI), a substance secreted by follicular cells. The
total number of primary oocytes at birth is estimated to vary from 700,000 to
2 million. During childhood most oocytes become atretic; only approximately
400,000 are present by the beginning of puberty, and fewer than 500 will be
ovulated. Some oocytes that reach maturity late in life have been dormant in
the diplotene stage of the ﬁrst meiotic division for 40 years or more before
ovulation. Whether the diplotene stage is the most suitable phase to protect
the oocyte against environmental inﬂuences is unknown. The fact that the risk
of having children with chromosomal abnormalities increases with maternal
age indicates that primary oocytes are vulnerable to damage as they age.
At puberty, a pool of growing follicles is established and continuously maintained from the supply of primordial follicles. Each month, 15 to 20 follicles
selected from this pool begin to mature, passing through three stages: 1) primary or preantral; 2) secondary or antral (also called vesicular or Graaﬁan);
and 3) preovulatory. The antral stage is the longest, whereas the preovulatory
stage encompasses approximately 37 hours before ovulation. As the primary
oocyte begins to grow, surrounding follicular cells change from ﬂat to cuboidal
and proliferate to produce a stratiﬁed epithelium of granulosa cells, and the unit
AF
Figure 1.19 A. Secondary (antral) stage follicle. The oocyte, surrounded by the zona
pellucida, is off-center; the antrum has developed by ﬂuid accumulation between intercellular spaces. Note the arrangement of cells of the theca interna and the theca
externa. B. Mature secondary (graaﬁan) follicle. The antrum has enlarged considerably,
is ﬁlled with follicular ﬂuid, and is surrounded by a stratiﬁed layer of granulosa cells.
The oocyte is embedded in a mound of granulosa cells, the cumulus oophorus. C. Photomicrograph of a mature secondary follicle with an enlarged ﬂuid-ﬁlled antrum (cavity,
Cav) and a diameter of 20 mm (×65). CO, cumulus oophorus; MG, granulosa cells; AF,
atretic follicle.
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
23
is called a primary follicle (Fig. 1.18, B and C ). Granulosa cells rest on a basement membrane separating them from surrounding stromal cells that form the
theca folliculi. Also, granulosa cells and the oocyte secrete a layer of glycoproteins on the surface of the oocyte, forming the zona pellucida (Fig. 1.18C ). As
follicles continue to grow, cells of the theca folliculi organize into an inner layer
of secretory cells, the theca interna, and an outer ﬁbrous capsule, the theca
externa. Also, small, ﬁnger-like processes of the follicular cells extend across
the zona pellucida and interdigitate with microvilli of the plasma membrane
of the oocyte. These processes are important for transport of materials from
follicular cells to the oocyte.
As development continues, ﬂuid-ﬁlled spaces appear between granulosa
cells. Coalescence of these spaces forms the antrum, and the follicle is termed
a secondary (vesicular, Graaﬁan) follicle. Initially, the antrum is crescent
shaped, but with time, it enlarges (Fig. 1.19). Granulosa cells surrounding the
oocyte remain intact and form the cumulus oophorus. At maturity, the secondary follicle may be 25 mm or more in diameter. It is surrounded by the
theca interna, which is composed of cells having characteristics of steroid secretion, rich in blood vessels, and the theca externa, which gradually merges
with the ovarian stroma (Fig. 1.19).
With each ovarian cycle, a number of follicles begin to develop, but usually only one reaches full maturity. The others degenerate and become atretic
(Fig. 1.19C ). When the secondary follicle is mature, a surge in luteinizing
hormone (LH) induces the preovulatory growth phase. Meiosis I is completed,
resulting in formation of two daughter cells of unequal size, each with 23 doublestructured chromosomes (Fig. 1.20, A and B). One cell, the secondary oocyte,
receives most of the cytoplasm; the other, the ﬁrst polar body, receives practically none. The ﬁrst polar body lies between the zona pellucida and the cell
Granulosa cells
Zona pellucida
A
Primary oocyte in division
B
Secondary oocyte and
polar body 1
Secondary oocyte
in division
C
Polar body in division
Figure 1.20 Maturation of the oocyte. A. Primary oocyte showing the spindle of the
ﬁrst meiotic division. B. Secondary oocyte and ﬁrst polar body. The nuclear membrane
is absent. C. Secondary oocyte showing the spindle of the second meiotic division. The
ﬁrst polar body is also dividing.
24
Part One: General Embryology
membrane of the secondary oocyte in the perivitelline space (Fig. 1.20B ). The
cell then enters meiosis II but arrests in metaphase approximately 3 hours
before ovulation. Meiosis II is completed only if the oocyte is fertilized; otherwise, the cell degenerates approximately 24 hours after ovulation. The ﬁrst
polar body also undergoes a second division (Fig. 1.20C).
SPERMATOGENESIS
Maturation of Sperm Begins at Puberty
Spermatogenesis, which begins at puberty, includes all of the events by which
spermatogonia are transformed into spermatozoa. At birth, germ cells in the
male can be recognized in the sex cords of the testis as large, pale cells surrounded by supporting cells (Fig. 1.21A). Supporting cells, which are derived
from the surface epithelium of the gland in the same manner as follicular cells,
become sustentacular cells, or Sertoli cells (Fig. 1.21C ).
Shortly before puberty, the sex cords acquire a lumen and become the
seminiferous tubules. At about the same time, primordial germ cells give
rise to spermatogonial stem cells. At regular intervals, cells emerge from this
stem cell population to form type A spermatogonia, and their production
marks the initiation of spermatogenesis. Type A cells undergo a limited number of mitotic divisions to form a clone of cells. The last cell division produces type B spermatogonia, which then divide to form primary spermatocytes (Figs. 1.21 and 1.22). Primary spermatocytes then enter a prolonged
Figure 1.21 A. Cross section through primitive sex cords of a newborn boy showing
primordial germ cells and supporting cells. B and C. Two segments of a seminiferous
tubule in transverse section. Note the different stages of spermatogenesis.
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
25
Type A dark
spermatogonia
Type A pale
spermatogonia
Type A pale
spermatogonia
Type A pale
spermatogonia
Type A pale
spermatogonia
Type B
spermatogonia
Primary
spermatocytes
Secondary
spermatocytes
Spermatids
Residual bodies
Spermatozoa
Figure 1.22 Type A spermatogonia, derived from the spermatogonial stem cell population, represent the ﬁrst cells in the process of spermatogenesis. Clones of cells are
established and cytoplasmic bridges join cells in each succeeding division until individual sperm are separated from residual bodies. In fact, the number of individual interconnected cells is considerably greater than depicted in this ﬁgure.
26
Part One: General Embryology
Type B
spermatogonium
Secondary
spermatocyte
Resting primary
spermatocyte
Spermatid
division
A
B
Mitotic
C
1st meiotic
division
D
2nd meiotic
division
Figure 1.23 The products of meiosis during spermatogenesis in humans.
prophase (22 days) followed by rapid completion of meiosis I and formation
of secondary spermatocytes. During the second meiotic division, these cells
immediately begin to form haploid spermatids (Figs. 1.21–1.23). Throughout
this series of events, from the time type A cells leave the stem cell population to formation of spermatids, cytokinesis is incomplete, so that successive
cell generations are joined by cytoplasmic bridges. Thus, the progeny of a single type A spermatogonium form a clone of germ cells that maintain contact
throughout differentiation (Fig. 1.22). Furthermore, spermatogonia and spermatids remain embedded in deep recesses of Sertoli cells throughout their
development (Fig. 1.24). In this manner, Sertoli cells support and protect the
germ cells, participate in their nutrition, and assist in the release of mature
spermatozoa.
Spermatogenesis is regulated by luteinizing hormone (LH) production by
the pituitary. LH binds to receptors on Leydig cells and stimulates testosterone
production, which in turn binds to Sertoli cells to promote spermatogenesis.
Follicle stimulating hormone (FSH) is also essential because its binding to
Sertoli cells stimulates testicular ﬂuid production and synthesis of intracellular
androgen receptor proteins.
Spermiogenesis
The series of changes resulting in the transformation of spermatids into spermatozoa is spermiogenesis. These changes include (a) formation of the acrosome,
which covers half of the nuclear surface and contains enzymes to assist in penetration of the egg and its surrounding layers during fertilization (Fig. 1.25);
(b) condensation of the nucleus; (c) formation of neck, middle piece, and tail;
and (d) shedding of most of the cytoplasm. In humans, the time required for
a spermatogonium to develop into a mature spermatozoon is approximately
64 days.
When fully formed, spermatozoa enter the lumen of seminiferous tubules.
From there, they are pushed toward the epididymis by contractile elements
in the wall of the seminiferous tubules. Although initially only slightly motile,
spermatozoa obtain full motility in the epididymis.
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
27
Late
spermatids
Early
spermatids
Primary
spermatocyte
Sertoli cell
Junctional
complex
Type A pale spermatogonia
Type A dark spermatogonia
Type B spermatogonia
Basal lamina
Peritubular cells
Figure 1.24 Sertoli cells and maturing spermatocytes. Spermatogonia, spermatocytes,
and early spermatids occupy depressions in basal aspects of the cell; late spermatids
are in deep recesses near the apex.
CLINICAL CORRELATES
Abnormal Gametes
In humans and in most mammals, one ovarian follicle occasionally contains
two or three clearly distinguishable primary oocytes (Fig. 1.26A). Although
these oocytes may give rise to twins or triplets, they usually degenerate before
reaching maturity. In rare cases, one primary oocyte contains two or even
three nuclei (Fig. 1.26B). Such binucleated or trinucleated oocytes die before
reaching maturity.
In contrast to atypical oocytes, abnormal spermatozoa are seen frequently, and up to 10% of all spermatozoa have observable defects. The
head or the tail may be abnormal; spermatozoa may be giants or dwarfs;
and sometimes they are joined (Fig. 1.26C ). Sperm with morphologic abnormalities lack normal motility and probably do not fertilize oocytes.
28
Part One: General Embryology
Figure 1.25 Important stages in transformation of the human spermatid into the spermatozoon.
Figure 1.26 Abnormal germ cells. A. Primordial follicle with two oocytes. B. Trinucleated oocyte. C. Various types of abnormal spermatozoa.
Summary
Primordial germ cells appear in the wall of the yolk sac in the fourth
week and migrate to the indifferent gonad (Fig. 1.1), where they arrive at the end of the ﬁfth week. In preparation for fertilization, both
male and female germ cells undergo gametogenesis, which includes meiosis and cytodifferentiation. During meiosis I, homologous chromosomes
pair and exchange genetic material; during meiosis II, cells fail to replicate
DNA, and each cell is thus provided with a haploid number of chromosomes
and half the amount of DNA of a normal somatic cell (Fig. 1.3). Hence, mature male and female gametes have, respectively, 22 plus X or 22 plus Y
chromosomes.
Birth defects may arise through abnormalities in chromosome number
or structure and from single gene mutations. Approximately 7% of major
Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes
29
birth defects are a result of chromosome abnormalities, and 8%, are a result of gene mutations. Trisomies (an extra chromosome) and monosomies
(loss of a chromosome) arise during mitosis or meiosis. During meiosis, homologous chromosomes normally pair and then separate. However, if separation fails (nondisjunction), one cell receives too many chromosomes and
one receives too few (Fig. 1.5). The incidence of abnormalities of chromosome number increases with age of the mother, particularly with mothers
aged 35 years and older. Structural abnormalities of chromosomes include
large deletions (cri-du-chat syndrome) and microdeletions. Microdeletions
involve contiguous genes that may result in defects such as Angelman syndrome (maternal deletion, chromosome 15q11–15q13) or Prader-Willi syndrome (paternal deletion, 15q11–15q13). Because these syndromes depend
on whether the affected genetic material is inherited from the mother or the
father, they also are an example of imprinting. Gene mutations may be dominant (only one gene of an allelic pair has to be affected to produce an alteration) or recessive (both allelic gene pairs must be mutated). Mutations responsible for many birth defects affect genes involved in normal embryological
development.
In the female, maturation from primitive germ cell to mature gamete, which
is called oogenesis, begins before birth; in the male, it is called spermatogenesis, and it begins at puberty. In the female, primordial germ cells form
oogonia. After repeated mitotic divisions, some of these arrest in prophase of
meiosis I to form primary oocytes. By the seventh month, nearly all oogonia have become atretic, and only primary oocytes remain surrounded by
a layer of follicular cells derived from the surface epithelium of the ovary
(Fig. 1.17). Together, they form the primordial follicle. At puberty, a pool of
growing follicles is recruited and maintained from the ﬁnite supply of primordial follicles. Thus, everyday 15 to 20 follicles begin to grow, and as they mature, they pass through three stages: 1) primary or preantral; 2) secondary
or antral (vesicular, Graaﬁan); and 3) preovulatory. The primary oocyte remains in prophase of the ﬁrst meiotic division until the secondary follicle is
mature. At this point, a surge in luteinizing hormone (LH) stimulates preovulatory growth: meiosis I is completed and a secondary oocyte and polar
body are formed. Then, the secondary oocyte is arrested in metaphase of
meiosis II approximately 3 hours before ovulation and will not complete this
cell division until fertilization. In the male, primordial cells remain dormant
until puberty, and only then do they differentiate into spermatogonia. These
stem cells give rise to primary spermatocytes, which through two successive
meiotic divisions produce four spermatids (Fig. 1.4). Spermatids go through
a series of changes (spermiogenesis) (Fig. 1.25) including (a) formation of
the acrosome, (b) condensation of the nucleus, (c) formation of neck, middle
piece, and tail, and (d) shedding of most of the cytoplasm. The time required
for a spermatogonium to become a mature spermatozoon is approximately
64 days.
30
Part One: General Embryology
Problems to Solve
1. What is the most common cause of abnormal chromosome number? Give an
example of a clinical syndrome involving abnormal numbers of chromosomes.
2. In addition to numerical abnormalities, what types of chromosomal
alterations occur?
3. What is mosaicism, and how does it occur?
SUGGESTED READING
Chandley AC: Meiosis in man. Trends Genet 4:79, 1988.
Clermont Y: Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 52:198, 1972.
Eddy EM, Clark JM, Gong D, Fenderson BA: Origin and migration of primordial germ cells in
mammals. Gamete Res 4:333, 1981.
Gelchrter TD, Collins FS: Principles of Medical Genetics. Baltimore, Williams & Wilkins, 1990.
Gorlin RJ, Cohen MM, Levin LS (eds): Syndromes of the Head and Neck. 3rd ed. New York, Oxford
University, 1990.
Heller CG, Clermont Y: Kinetics of the germinal epithelium in man. Recent Prog Horm Res 20:545,
1964.
Johnson MH, Everett BJ: Essential Reproduction. 5th ed. London, Blackwell Science Limited, 2000.
Jones KL (ed): Smith’s Recognizable Patterns of Human Malformation. 4th ed. Philadelphia, WB
Saunders, 1988.
Larsen WJ, Wert SE: Roles of cell junctions in gametogenesis and early embryonic development.
Tissue Cell 20:809, 1988.
Lenke RR, Levy HL: Maternal phenylketonuria and hyperphenylalaninemia: an international survey
of untreated and treated pregnancies. N Engl J Med 303:1202, 1980.
Pelletier RA, We K, Balakier H: Development of membrane differentiations in the guinea pig spermatid during spermiogenesis. Am J Anat 167:119, 1983.
Russell LD: Sertoligerm cell interactions: a review. Gamete Res 3:179, 1980.
Stevenson RE, Hall JG, Goodman RM (eds): Human Malformations and Related Anomalies. Vol I, II.
New York, Oxford University Press, 1993.
Thorogood P (ed): Embryos, Genes, and Birth Defects. New York, Wiley, 1997.
Witschj E: Migration of the germ cells of the human embryos from the yolk sac to the primitive
gonadal folds. Contrib Embryol 36:67, 1948.
c h a p t e r
2
First Week of Development:
Ovulation to Implantation
Ovarian Cycle
At puberty, the female begins to undergo regular
monthly cycles. These sexual cycles are controlled
by the hypothalamus. Gonadotropin-releasing hormone (GnRH) produced by the hypothalamus acts on
cells of the anterior pituitary gland, which in turn secrete
gonadotropins. These hormones, follicle-stimulating
hormone (FSH) and luteinizing hormone (LH), stimulate
and control cyclic changes in the ovary.
At the beginning of each ovarian cycle, 15 to 20 primary
(preantral) stage follicles are stimulated to grow under the
inﬂuence of FSH. (The hormone is not necessary to promote
development of primordial follicles to the primary follicle stage,
but without it, these primary follicles die and become atretic.) Thus,
FSH rescues 15 to 20 of these cells from a pool of continuously
forming primary follicles (Fig. 2.1). Under normal conditions, only
one of these follicles reaches full maturity, and only one oocyte is
discharged; the others degenerate and become atretic. In the next
cycle, another group of primary follicles is recruited, and again, only
one follicle reaches maturity. Consequently, most follicles degenerate
without ever reaching full maturity. When a follicle becomes atretic,
the oocyte and surrounding follicular cells degenerate and are replaced
by connective tissue, forming a corpus atreticum. FSH also stimulates
maturation of follicular (granulosa) cells surrounding the oocyte. In
turn, proliferation of these cells is mediated by growth differentiation
31
32
Part One: General Embryology
Primary oocyte
Granulosa
cells
Zona pellucida
Theca
externa
Antrum
Theca
interna
Primordial follicle
Primary follicle
Secondary follicle
Figure 2.1 From the pool of primordial follicles, every day some begin to grow and develop into secondary (preantral) follicles, and this growth is independent of FSH. Then,
as the cycle progresses, FSH secretion recruits primary follicles to begin development
into secondary (antral, Graaﬁan) follicles. During the last few days of maturation of secondary follicles, estrogens, produced by follicular and thecal cells, stimulate increased
production of LH by the pituitary (Fig. 2.13), and this hormone causes the follicle to
enter the preovulatory stage, to complete meiosis I, and to enter meiosis II where it
arrests in metaphase approximately 3 hours before ovulation.
factor-9 (GDF-9), a member of the transforming growth factor-β (TGF-β) family.
In cooperation, granulosa and thecal cells produce estrogens that (a) cause the
uterine endometrium to enter the follicular or proliferative phase; (b) cause
thinning of the cervical mucus to allow passage of sperm; and (c) stimulate the
pituitary gland to secrete LH. At mid-cycle, there is an LH surge that (a) elevates concentrations of maturation-promoting factor, causing oocytes to complete meiosis I and initiate meiosis II; (b) stimulates production of progesterone
by follicular stromal cells (luteinization); and (c) causes follicular rupture and
ovulation.
OVULATION
In the days immediately preceding ovulation, under the inﬂuence of FSH and
LH, the secondary follicle grows rapidly to a diameter of 25 mm. Coincident
with ﬁnal development of the secondary follicle, there is an abrupt increase in
LH that causes the primary oocyte to complete meiosis I and the follicle to enter
the preovulatory stage. Meiosis II is also initiated, but the oocyte is arrested in
metaphase approximately 3 hours before ovulation. In the meantime, the surface of the ovary begins to bulge locally, and at the apex, an avascular spot, the
stigma, appears. The high concentration of LH increases collagenase activity,
resulting in digestion of collagen ﬁbers surrounding the follicle. Prostaglandin
levels also increase in response to the LH surge and cause local muscular contractions in the ovarian wall. Those contractions extrude the oocyte, which
together with its surrounding granulosa cells from the region of the cumulus
Chapter 2: First Week of Development: Ovulation to Implantation
Antrum
Granulosa cells
Theca interna
Oocyte in
2nd meiotic
division
Preovulatory follicle
Luteal cells
Ovarian stroma
Theca
externa
Blood
vessels
1st
polar
body
A
33
B
Cumulus oophorus Fibrin
cells
Ovulation
C Corpus luteum
Figure 2.2 A. Preovulatory follicle bulging at the ovarian surface. B. Ovulation. The
oocyte, in metaphase of meiosis II, is discharged from the ovary together with a large
number of cumulus oophorus cells. Follicular cells remaining inside the collapsed follicle differentiate into lutean cells. C. Corpus luteum. Note the large size of the corpus
luteum, caused by hypertrophy and accumulation of lipid in granulosa and theca interna
cells. The remaining cavity of the follicle is ﬁlled with ﬁbrin.
oophorus, breaks free (ovulation) and ﬂoats out of the ovary (Figs. 2.2 and
2.3). Some of the cumulus oophorus cells then rearrange themselves around
the zona pellucida to form the corona radiata (Figs. 2.4–2.6).
CLINICAL CORRELATES
Ovulation
During ovulation, some women feel a slight pain, known as middle pain
because it normally occurs near the middle of the menstrual cycle. Ovulation
is also generally accompanied by a rise in basal temperature, which can be
monitored to aid in determining when release of the oocyte occurs. Some
women fail to ovulate because of a low concentration of gonadotropins. In
these cases, administration of an agent to stimulate gonadotropin release and
hence ovulation can be employed. Although such drugs are effective, they
often produce multiple ovulations, so that the risk of multiple pregnancies is
10 times higher in these women than in the general population.
CORPUS LUTEUM
After ovulation, granulosa cells remaining in the wall of the ruptured follicle,
together with cells from the theca interna, are vascularized by surrounding vessels. Under the inﬂuence of LH, these cells develop a yellowish pigment and
change into lutean cells, which form the corpus luteum and secrete the hormone progesterone (Fig. 2.2C ). Progesterone, together with estrogenic hormones, causes the uterine mucosa to enter the progestational or secretory
stage in preparation for implantation of the embryo.
34
Part One: General Embryology
Figure 2.3 A. Scanning electron micrograph of ovulation in the mouse. The surface
of the oocyte is covered by the zona pellucida. The cumulus oophorus is composed of
granulosa cells. B. Scanning electron micrograph of a rabbit oocyte 1.5 hours after
ovulation. The oocyte, which is surrounded by granulosa cells, lies on the surface of the
ovary. Note the site of ovulation.
Chapter 2: First Week of Development: Ovulation to Implantation
35
Figure 2.4 Relation of ﬁmbriae and ovary. Fimbriae collect the oocyte and sweep it into
the uterine tube.
OOCYTE TRANSPORT
Shortly before ovulation, ﬁmbriae of the oviduct begin to sweep over the surface
of the ovary, and the tube itself begins to contract rhythmically. It is thought that
the oocyte surrounded by some granulosa cells (Figs. 2.3 and 2.4) is carried
into the tube by these sweeping movements of the ﬁmbriae and by motion
of cilia on the epithelial lining. Once in the tube, cumulus cells withdraw their
cytoplasmic processes from the zona pellucida and lose contact with the oocyte.
Once the oocyte is in the uterine tube, it is propelled by cilia with the rate
of transport regulated by the endocrine status during and after ovulation. In
humans, the fertilized oocyte reaches the uterine lumen in approximately 3 to
4 days.
CORPUS ALBICANS
If fertilization does not occur, the corpus luteum reaches maximum development approximately 9 days after ovulation. It can easily be recognized as a yellowish projection on the surface of the ovary. Subsequently, the corpus luteum
shrinks because of degeneration of lutean cells and forms a mass of ﬁbrotic
36
Part One: General Embryology
A
B
Figure 2.5 A. Scanning electron micrograph of sperm binding to the zona pellucida.
B. The three phases of oocyte penetration. In phase 1, spermatozoa pass through the
corona radiata barrier; in phase 2, one or more spermatozoa penetrate the zona pellucida; in phase 3, one spermatozoon penetrates the oocyte membrane while losing its
own plasma membrane. Inset. Normal spermatocyte with acrosomal head cap.
Chapter 2: First Week of Development: Ovulation to Implantation
37
Figure 2.6 A. Oocyte immediately after ovulation, showing the spindle of the second
meiotic division. B. A spermatozoon has penetrated the oocyte, which has ﬁnished its
second meiotic division. Chromosomes of the oocyte are arranged in a vesicular nucleus,
the female pronucleus. Heads of several sperm are stuck in the zona pellucida. C. Male
and female pronuclei. D and E. Chromosomes become arranged on the spindle, split
longitudinally, and move to opposite poles. F. Two-cell stage.
scar tissue, the corpus albicans. Simultaneously, progesterone production decreases, precipitating menstrual bleeding. If the oocyte is fertilized, degeneration of the corpus luteum is prevented by human chorionic gonadotropin
(hCG), a hormone secreted by the syncytiotrophoblast of the developing embryo. The corpus luteum continues to grow and forms the corpus luteum of
pregnancy (corpus luteum graviditatis). By the end of the third month, this
structure may be one-third to one-half of the total size of the ovary. Yellowish
luteal cells continue to secrete progesterone until the end of the fourth month;
thereafter, they regress slowly as secretion of progesterone by the trophoblastic
component of the placenta becomes adequate for maintenance of pregnancy.
Removal of the corpus luteum of pregnancy before the fourth month usually
leads to abortion.
Fertilization
Fertilization, the process by which male and female gametes fuse, occurs in the
ampullary region of the uterine tube. This is the widest part of the tube and
38
Part One: General Embryology
is close to the ovary (Fig. 2.4). Spermatozoa may remain viable in the female
reproductive tract for several days.
Only 1% of sperm deposited in the vagina enter the cervix, where they
may survive for many hours. Movement of sperm from the cervix to the oviduct
is accomplished primarily by their own propulsion, although they may be assisted by movements of ﬂuids created by uterine cilia. The trip from cervix
to oviduct requires a minimum of 2 to 7 hours, and after reaching the isthmus, sperm become less motile and cease their migration. At ovulation, sperm
again become motile, perhaps because of chemoattractants produced by cumulus cells surrounding the egg, and swim to the ampulla where fertilization
usually occurs. Spermatozoa are not able to fertilize the oocyte immediately
upon arrival in the female genital tract but must undergo (a) capacitation and
(b) the acrosome reaction to acquire this capability.
Capacitation is a period of conditioning in the female reproductive tract
that in the human lasts approximately 7 hours. Much of this conditioning,
which occurs in the uterine tube, entails epithelial interactions between the
sperm and mucosal surface of the tube. During this time a glycoprotein coat
and seminal plasma proteins are removed from the plasma membrane that
overlies the acrosomal region of the spermatozoa. Only capacitated sperm can
pass through the corona cells and undergo the acrosome reaction.
The acrosome reaction, which occurs after binding to the zona pellucida,
is induced by zona proteins. This reaction culminates in the release of enzymes
needed to penetrate the zona pellucida, including acrosin and trypsin-like substances (Fig. 2.5).
The phases of fertilization include phase 1, penetration of the corona radiata; phase 2, penetration of the zona pellucida; and phase 3, fusion of the
oocyte and sperm cell membranes.
PHASE 1: PENETRATION OF THE CORONA RADIATA
Of the 200 to 300 million spermatozoa deposited in the female genital tract,
only 300 to 500 reach the site of fertilization. Only one of these fertilizes the
egg. It is thought that the others aid the fertilizing sperm in penetrating the
barriers protecting the female gamete. Capacitated sperm pass freely through
corona cells (Fig. 2.5).
PHASE 2: PENETRATION OF THE ZONA PELLUCIDA
The zona is a glycoprotein shell surrounding the egg that facilitates and maintains sperm binding and induces the acrosome reaction. Both binding and the
acrosome reaction are mediated by the ligand ZP3, a zona protein. Release
of acrosomal enzymes (acrosin) allows sperm to penetrate the zona, thereby
coming in contact with the plasma membrane of the oocyte (Fig. 2.5). Permeability of the zona pellucida changes when the head of the sperm comes
in contact with the oocyte surface. This contact results in release of lysosomal
Chapter 2: First Week of Development: Ovulation to Implantation
39
enzymes from cortical granules lining the plasma membrane of the oocyte.
In turn, these enzymes alter properties of the zona pellucida (zona reaction)
to prevent sperm penetration and inactivate species-speciﬁc receptor sites for
spermatozoa on the zona surface. Other spermatozoa have been found embedded in the zona pellucida, but only one seems to be able to penetrate the oocyte
(Fig. 2.6).
PHASE 3: FUSION OF THE OOCYTE AND
SPERM CELL MEMBRANES
The initial adhesion of sperm to the oocyte is mediated in part by the interaction of integrins on the oocyte and their ligands, disintegrins, on sperm. After
adhesion, the plasma membranes of the sperm and egg fuse (Fig. 2.5). Because
the plasma membrane covering the acrosomal head cap disappears during the
acrosome reaction, actual fusion is accomplished between the oocyte membrane and the membrane that covers the posterior region of the sperm head
(Fig. 2.5). In the human, both the head and tail of the spermatozoon enter the
cytoplasm of the oocyte, but the plasma membrane is left behind on the oocyte
surface. As soon as the spermatozoon has entered the oocyte, the egg responds
in three ways:
1. Cortical and zona reactions. As a result of the release of cortical oocyte
granules, which contain lysosomal enzymes, (a) the oocyte membrane
becomes impenetrable to other spermatozoa, and (b) the zona pellucida alters its structure and composition to prevent sperm binding and
penetration. These reactions prevent polyspermy (penetration of more
than one spermatozoon into the oocyte).
2. Resumption of the second meiotic division. The oocyte ﬁnishes its second meiotic division immediately after entry of the spermatozoon. One
of the daughter cells, which receives hardly any cytoplasm, is known as
the second polar body; the other daughter cell is the deﬁnitive oocyte.
Its chromosomes (22 + X) arrange themselves in a vesicular nucleus
known as the female pronucleus (Figs. 2.6 and 2.7).
3. Metabolic activation of the egg. The activating factor is probably carried by the spermatozoon. Postfusion activation may be considered to
encompass the initial cellular and molecular events associated with early
embryogenesis.
The spermatozoon, meanwhile, moves forward until it lies close to the
female pronucleus. Its nucleus becomes swollen and forms the male pronucleus (Fig. 2.6); the tail detaches and degenerates. Morphologically, the male
and female pronuclei are indistinguishable, and eventually, they come into
close contact and lose their nuclear envelopes (Fig. 2.7A). During growth of
male and female pronuclei (both haploid), each pronucleus must replicate its
DNA. If it does not, each cell of the two-cell zygote has only half of the normal
amount of DNA. Immediately after DNA synthesis, chromosomes organize on
40
Part One: General Embryology
Figure 2.7 A. Phase contrast view of the pronuclear stage of a fertilized human oocyte
with male and female pronuclei. B. Two-cell stage of human zygote.
the spindle in preparation for a normal mitotic division. The 23 maternal and
23 paternal (double) chromosomes split longitudinally at the centromere, and
sister chromatids move to opposite poles, providing each cell of the zygote
with the normal diploid number of chromosomes and DNA (Fig. 2.6, D and
E ). As sister chromatids move to opposite poles, a deep furrow appears on the
surface of the cell, gradually dividing the cytoplasm into two parts (Figs. 2.6F
and 2.7B ).
The main results of fertilization are as follows:
r Restoration of the diploid number of chromosomes, half from the fa-
ther and half from the mother. Hence, the zygote contains a new combination of chromosomes different from both parents.
r Determination of the sex of the new individual. An X-carrying sperm
r
produces a female (XX) embryo, and a Y-carrying sperm produces a male
(XY) embryo. Hence, the chromosomal sex of the embryo is determined
at fertilization.
Initiation of cleavage. Without fertilization, the oocyte usually degenerates 24 hours after ovulation.
CLINICAL CORRELATES
Contraceptive Methods
Barrier techniques of contraception include the male condom, made of latex
and often containing chemical spermicides, which ﬁts over the penis; and
the female condom, made of polyurethane, which lines the vagina. Other
barriers placed in the vagina include the diaphragm, the cervical cap, and the
contraceptive sponge.
The contraceptive pill is a combination of estrogen and the progesterone
analogue progestin, which together inhibit ovulation but permit menstruation.
Chapter 2: First Week of Development: Ovulation to Implantation
41
Both hormones act at the level of FSH and LH, preventing their release from
the pituitary. The pills are taken for 21 days and then stopped to allow menstruation, after which the cycle is repeated.
Depo-Provera is a progestin compound that can be implanted subdermally or injected intramuscularly to prevent ovulation for up to 5 years or 23
months, respectively.
A male “pill” has been developed and tested in clinical trials. It contains a
synthetic androgen that prevents both LH and FSH secretion and either stops
sperm production (70–90% of men) or reduces it to a level of infertility.
The intrauterine device (IUD) is placed in the uterine cavity. Its mechanism for preventing pregnancy is not clear but may entail direct effects on
sperm and oocytes or inhibition of preimplantation stages of development.
The drug RU-486 (mifepristone) causes abortion if it is administered
within 8 weeks of the previous menses. It initiates menstruation, possibly
through its action as an antiprogesterone agent.
Vasectomy and tubal ligation are effective means of contraception, and
both procedures are reversible, although not in every case.
Infertility
Infertility is a problem for 15% to 30% of couples. Male infertility may be
a result of insufﬁcient numbers of sperm and/or poor motility. Normally, the
ejaculate has a volume of 3 to 4 ml, with approximately 100 million sperm
per ml. Males with 20 million sperm per ml or 50 million sperm per total
ejaculate are usually fertile. Infertility in a woman may be due to a number of
causes, including occluded oviducts (most commonly caused by pelvic inﬂammatory disease), hostile cervical mucus, immunity to spermatozoa, absence
of ovulation, and others.
In vitro fertilization (IVF) of human ova and embryo transfer is a frequent
practice conducted by laboratories throughout the world. Follicle growth in the
ovary is stimulated by administration of gonadotropins. Oocytes are recovered
by laparoscopy from ovarian follicles with an aspirator just before ovulation
when the oocyte is in the late stages of the ﬁrst meiotic division. The egg is
placed in a simple culture medium and sperm are added immediately. Fertilized eggs are monitored to the eight-cell stage and then placed in the uterus
to develop to term. Fortunately, because preimplantation-stage embryos are
resistant to teratogenic insult, the risk of producing malformed offspring by
in vitro procedures is low.
A disadvantage of IVF is its low success rate; only 20% of fertilized ova
implant and develop to term. Therefore, to increase chances of a successful
pregnancy, four or ﬁve ova are collected, fertilized, and placed in the uterus.
This approach sometimes leads to multiple births.
Another technique, gamete intrafallopian transfer (GIFT), introduces
oocytes and sperm into the ampulla of the fallopian (uterine) tube, where
42
Part One: General Embryology
fertilization takes place. Development then proceeds in a normal fashion. In a
similar approach, zygote intrafallopian transfer (ZIFT), fertilized oocytes are
placed in the ampullary region. Both of these methods require patent uterine
tubes.
Severe male infertility, in which the ejaculate contains very few live sperm
(oligozoospermia) or even no live sperm (azoospermia), can be overcome
using intracytoplasmic sperm injection (ICSI). With this technique, a single
sperm, which may be obtained from any point in the male reproductive tract,
is injected into the cytoplasm of the egg to cause fertilization. This approach
offers couples an alternative to using donor sperm for IVF. The technique
carries an increased risk for fetuses to have Y chromosome deletions but no
other chromosomal abnormalities.
Cleavage
Once the zygote has reached the two-cell stage, it undergoes a series of mitotic
divisions, increasing the numbers of cells. These cells, which become smaller
with each cleavage division, are known as blastomeres (Fig. 2.8). Until the
eight-cell stage, they form a loosely arranged clump (Fig. 2.9A). However, after
the third cleavage, blastomeres maximize their contact with each other, forming a compact ball of cells held together by tight junctions (Fig. 2.9B). This
process, compaction, segregates inner cells, which communicate extensively
by gap junctions, from outer cells. Approximately 3 days after fertilization, cells
of the compacted embryo divide again to form a 16-cell morula (mulberry).
Inner cells of the morula constitute the inner cell mass, and surrounding cells
compose the outer cell mass. The inner cell mass gives rise to tissues of the
Figure 2.8 Development of the zygote from the two-cell stage to the late morula
stage. The two-cell stage is reached approximately 30 hours after fertilization; the
four-cell stage, at approximately 40 hours; the 12- to 16-cell stage, at approximately
3 days; and the late morula stage, at approximately 4 days. During this period, blastomeres are surrounded by the zona pellucida, which disappears at the end of the fourth
day.
Chapter 2: First Week of Development: Ovulation to Implantation
A
43
B
Figure 2.9 Scanning electron micrographs of uncompacted (A) and compacted (B)
eight-cell mouse embryos. In the uncompacted state, outlines of each blastomere are
distinct, whereas after compaction cell-cell contacts are maximized and cellular outlines
are indistinct.
embryo proper, and the outer cell mass forms the trophoblast, which later
contributes to the placenta.
Blastocyst Formation
About the time the morula enters the uterine cavity, ﬂuid begins to penetrate
through the zona pellucida into the intercellular spaces of the inner cell mass.
Gradually the intercellular spaces become conﬂuent, and ﬁnally a single cavity,
the blastocele, forms (Fig. 2.10, A and B ). At this time, the embryo is a
blastocyst. Cells of the inner cell mass, now called the embryoblast, are at
one pole, and those of the outer cell mass, or trophoblast, ﬂatten and form
the epithelial wall of the blastocyst (Fig. 2.10, A and B). The zona pellucida
has disappeared, allowing implantation to begin.
In the human, trophoblastic cells over the embryoblast pole begin to penetrate between the epithelial cells of the uterine mucosa about the sixth day
(Fig. 2.10C ). Attachment and invasion of the trophoblast involve integrins, expressed by the trophoblast, and the extracellular matrix molecules laminin and
ﬁbronectin. Integrin receptors for laminin promote attachment, while those
for ﬁbronectin stimulate migration. These molecules also interact along signal
transduction pathways to regulate trophoblast differentiation so that implantation is the result of mutual trophoblastic and endometrial action. Hence, by the
end of the ﬁrst week of development, the human zygote has passed through
the morula and blastocyst stages and has begun implantation in the uterine
mucosa.
44
Part One: General Embryology
A
Figure 2.10 A. Section of a 107-cell human blastocyst showing inner cell mass and
trophoblast cells. B. Schematic representation of a human blastocyst recovered from the
uterine cavity at approximately 4.5 days. Blue, inner cell mass or embryoblast; green,
trophoblast. C. Schematic representation of a blastocyst at the ninth day of development
showing trophoblast cells at the embryonic pole of the blastocyst penetrating the uterine
mucosa. The human blastocyst begins to penetrate the uterine mucosa by the sixth day
of development.
CLINICAL CORRELATES
Abnormal Zygotes
The exact number of abnormal zygotes formed is unknown because they
are usually lost within 2 to 3 weeks of fertilization, before the woman realizes she is pregnant, and therefore are not detected. Estimates are that
as many as 50% of pregnancies end in spontaneous abortion and that
Chapter 2: First Week of Development: Ovulation to Implantation
45
half of these losses are a result of chromosomal abnormalities. These abortions
are a natural means of screening embryos for defects, reducing the incidence
of congenital malformations. Without this phenomenon, approximately 12%
instead of 2% to 3% of infants would have birth defects.
With the use of a combination of IVF and polymerase chain reaction
(PCR), molecular screening of embryos for genetic defects is being conducted.
Single blastomeres from early-stage embryos can be removed and their DNA
ampliﬁed for analysis. As the Human Genome Project provides more sequencing information and as speciﬁc genes are linked to various syndromes, such
procedures will become more commonplace.
Uterus at Time of Implantation
The wall of the uterus consists of three layers: (a) endometrium or mucosa lining the inside wall; (b) myometrium, a thick layer of smooth muscle; and (c) perimetrium, the peritoneal covering lining the outside wall
(Fig. 2.11). From puberty (11–13 years) until menopause (45–50 years), the
endometrium undergoes changes in a cycle of approximately 28 days under
hormonal control by the ovary. During this menstrual cycle, the uterine endometrium passes through three stages, the follicular or proliferative phase,
Preovulatory
Figure 2.11 Events during the ﬁrst week of human development. 1, Oocyte immediately after ovulation. 2, Fertilization, approximately 12 to 24 hours after ovulation. 3,
Stage of the male and female pronuclei. 4, Spindle of the ﬁrst mitotic division. 5, Twocell stage (approximately 30 hours of age). 6, Morula containing 12 to 16 blastomeres
(approximately 3 days of age). 7, Advanced morula stage reaching the uterine lumen
(approximately 4 days of age). 8, Early blastocyst stage (approximately 4.5 days of age).
The zona pellucida has disappeared. 9, Early phase of implantation (blastocyst approximately 6 days of age). The ovary shows stages of transformation between a primary
follicle and a preovulatory follicle as well as a corpus luteum. The uterine endometrium
is shown in the progestational stage.
46
Part One: General Embryology
Maturation of follicle
Ovulation
Corpus luteum
Corpus luteum
of pregnancy
Implanted embryo
Implantation begins
Gland
Compact layer
Spongy layer
Basal
layer
4
0
Menstrual phase
14
Follicular or
proliferative phase
28
Progestational or
secretory phase
Gravid phase
Figure 2.12 Changes in the uterine mucosa correlated with those in the ovary. Implantation of the blastocyst has caused development of a large corpus luteum of pregnancy.
Secretory activity of the endometrium increases gradually as a result of large amounts
of progesterone produced by the corpus luteum of pregnancy.
the secretory or progestational phase, and the menstrual phase (Figs. 2.11–
2.13). The proliferative phase begins at the end of the menstrual phase, is under
the inﬂuence of estrogen, and parallels growth of the ovarian follicles. The secretory phase begins approximately 2 to 3 days after ovulation in response to
progesterone produced by the corpus luteum. If fertilization does not occur,
shedding of the endometrium (compact and spongy layers) marks the beginning of the menstrual phase. If fertilization does occur, the endometrium assists
in implantation and contributes to formation of the placenta.
At the time of implantation, the mucosa of the uterus is in the secretory
phase (Figs. 2.11 and 2.12), during which time uterine glands and arteries
become coiled and the tissue becomes succulent. As a result, three distinct
layers can be recognized in the endometrium: a superﬁcial compact layer, an
intermediate spongy layer, and a thin basal layer (Fig. 2.12). Normally, the
human blastocyst implants in the endometrium along the anterior or posterior
wall of the body of the uterus, where it becomes embedded between the
openings of the glands (Fig. 2.12).
If the oocyte is not fertilized, venules and sinusoidal spaces gradually become packed with blood cells, and an extensive diapedesis of blood into the
tissue is seen. When the menstrual phase begins, blood escapes from superﬁcial arteries, and small pieces of stroma and glands break away. During the
following 3 or 4 days, the compact and spongy layers are expelled from the
Chapter 2: First Week of Development: Ovulation to Implantation
47
Figure 2.13 Changes in the uterine mucosa (endometrium) and corresponding changes
in the ovary during a regular menstrual cycle without fertilization.
uterus, and the basal layer is the only part of the endometrium that is retained
(Fig. 2.13). This layer, which is supplied by its own arteries, the basal arteries,
functions as the regenerative layer in the rebuilding of glands and arteries in
the proliferative phase (Fig. 2.13).
Summary
With each ovarian cycle, a number of primary follicles begin to grow, but
usually only one reaches full maturity, and only one oocyte is discharged
at ovulation. At ovulation, the oocyte is in metaphase of the second
meiotic division and is surrounded by the zona pellucida and some granulosa
cells (Fig. 2.4). Sweeping action of tubal ﬁmbriae carries the oocyte into the
uterine tube.
48
Part One: General Embryology
Before spermatozoa can fertilize the oocyte, they must undergo (a) capacitation, during which time a glycoprotein coat and seminal plasma proteins
are removed from the spermatozoon head, and (b) the acrosome reaction,
during which acrosin and trypsin-like substances are released to penetrate the
zona pellucida. During fertilization, the spermatozoon must penetrate (a) the
corona radiata, (b) the zona pellucida, and (c) the oocyte cell membrane
(Fig. 2.5). As soon as the spermatocyte has entered the oocyte, (a) the oocyte
ﬁnishes its second meiotic division and forms the female pronucleus; (b) the
zona pellucida becomes impenetrable to other spermatozoa; and (c) the head
of the sperm separates from the tail, swells, and forms the male pronucleus
(Figs. 2.6 and 2.7). After both pronuclei have replicated their DNA, paternal
and maternal chromosomes intermingle, split longitudinally, and go through a
mitotic division, giving rise to the two-cell stage. The results of fertilization are
(a) restoration of the diploid number of chromosomes, (b) determination of
chromosomal sex, and (c) initiation of cleavage.
Cleavage is a series of mitotic divisions that results in an increase in cells,
blastomeres, which become smaller with each division. After three divisions,
blastomeres undergo compaction to become a tightly grouped ball of cells with
inner and outer layers. Compacted blastomeres divide to form a 16-cell morula.
As the morula enters the uterus on the third or fourth day after fertilization, a
cavity begins to appear, and the blastocyst forms. The inner cell mass, which
is formed at the time of compaction and will develop into the embryo proper,
is at one pole of the blastocyst. The outer cell mass, which surrounds the inner
cells and the blastocyst cavity, will form the trophoblast.
The uterus at the time of implantation is in the secretory phase, and the
blastocyst implants in the endometrium along the anterior or posterior wall. If
fertilization does not occur, then the menstrual phase begins and the spongy
and compact endometrial layers are shed. The basal layer remains to regenerate
the other layers during the next cycle.
Problems to Solve
1. What are the primary causes of infertility in men and women?
2. A woman has had several bouts of pelvic inﬂammatory disease and now wants
to have children. However, she has been having difﬁculty becoming pregnant.
What is likely to be the problem, and what would you suggest?
SUGGESTED READING
Allen CA, Green DPL: The mammalian acrosome reaction: gateway to sperm fusion with the oocyte?
Bioessays 19:241, 1997.
Archer DF, Zeleznik AJ, Rockette HE: Ovarian follicular maturation in women: 2. Reversal of estrogen
inhibited ovarian folliculogenesis by human gonadotropin. Fertil Steril 50:555, 1988.
Barratt CLR, Cooke ID: Sperm transport in the human female reproductive tract: a dynamic interaction. Int J Androl 14:394, 1991.
Chapter 2: First Week of Development: Ovulation to Implantation
49
Boldt J, et al: Carbohydrate involvement in sperm-egg fusion in mice. Biol Reprod 40:887, 1989.
Burrows TD, King A, Loke YW: Expression of integrins by human trophoblast and differential
adhesion to laminin or ﬁbronectin. Hum Reprod 8:475, 1993.
Carr DH: Chromosome studies on selected spontaneous abortions: polyploidy in man. J Med Genet
8:164, 1971.
Chen CM, Sathananthan AH: Early penetration of human sperm through the vestments of human
egg in vitro. Arch Androl 16:183, 1986.
Cowchock S: Autoantibodies and fetal wastage. Am J Reprod Immunol 26:38, 1991.
Edwards RG: A decade of in vitro fertilization. Res Reprod 22:1, 1990.
Edwards RG, Bavister BD, Steptoe PC: Early stages of fertilization in vitro of human oocytes matured
in vitro. Nature (Lond) 221:632, 1969.
Egarter C: The complex nature of egg transport through the oviduct. Am J Obstet Gynecol 163:687,
1990.
Enders AC, Hendrickx AG, Schlake S: Implantation in the rhesus monkey: initial penetration of the
endometrium. Am J Anat 167:275, 1983.
Gilbert SF: Developmental Biology. Sunderland, MA, Sinauer, 1991.
Handyside AH, Kontogianni EH, Hardy K, Winston RML: Pregnancies from biopsied human preimplantation embryos sexed by Y-speciﬁc DNA ampliﬁcation. Nature 344:768, 1990.
Hertig AT, Rock J, Adams EC: A description of 34 human ova within the ﬁrst 17 days of development.
Am J Anat 98:435, 1956.
Johnson MH, Everitt BJ: Essential Reproduction. 5th ed. London, Blackwell Science Limited, 2000.
Liu J, et al: Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm
injection cycles. Hum Reprod 10:2630, 1995.
Oura C, Toshimori K: Ultrasound studies on the fertilization of mammalian gametes. Rev Cytol
122:105, 1990.
Pedersen RA, We K, Balakier H: Origin of the inner cell mass in mouse embryos: cell lineage
analysis by microinjection. Dev Biol 117:581, 1986.
Reproduction (entire issue). J NIH Res 9:1997.
Scott RT, Hodgen GD: The ovarian follicle: life cycle of a pelvic clock. Clin Obstet Gynecol 33:551,
1990.
Wasserman PM: Fertilization in mammals. Sci Am 259:78, 1988.
Wolf DP, Quigley MM (eds): Human in Vitro Fertilization and Transfer. New York, Plenum, 1984.
Yen SC, Jaffe RB (eds): Reproductive Endocrinology: Physiology, Pathophysiology, and Clinical Management. 2nd ed. Philadelphia, WB Saunders, 1986.
c h a p t e r
3
Second Week of Development:
Bilaminar Germ Disc
This chapter gives a day-by-day account of the
major events of the second week of development.
However, embryos of the same fertilization age do
not necessarily develop at the same rate. Indeed, considerable differences in rate of growth have been found
even at these early stages of development.
Day 8
At the eighth day of development, the blastocyst is partially
embedded in the endometrial stroma. In the area over the embryoblast, the trophoblast has differentiated into two layers:
(a) an inner layer of mononucleated cells, the cytotrophoblast,
and (b) an outer multinucleated zone without distinct cell boundaries, the syncytiotrophoblast (Figs. 3.1 and 3.2). Mitotic ﬁgures are
found in the cytotrophoblast but not in the syncytiotrophoblast. Thus,
cells in the cytotrophoblast divide and migrate into the syncytiotrophoblast, where they fuse and lose their individual cell membranes.
Cells of the inner cell mass or embryoblast also differentiate into two
layers: (a) a layer of small cuboidal cells adjacent to the blastocyst cavity,
known as the hypoblast layer, and (b) a layer of high columnar cells
adjacent to the amniotic cavity, the epiblast layer (Figs. 3.1 and 3.2).
Together, the layers form a ﬂat disc. At the same time, a small cavity
appears within the epiblast. This cavity enlarges to become the
51
52
Part One: General Embryology
Figure 3.1 A 7.5-day human blastocyst, partially embedded in the endometrial stroma.
The trophoblast consists of an inner layer with mononuclear cells, the cytotrophoblast,
and an outer layer without distinct cell boundaries, the syncytiotrophoblast. The embryoblast is formed by the epiblast and hypoblast layers. The amniotic cavity appears
as a small cleft.
Figure 3.2 Section of a 7.5-day human blastocyst (×100). Note the multinucleated appearance of the syncytiotrophoblast, large cells of the cytotrophoblast, and slit-like
amniotic cavity.
amniotic cavity. Epiblast cells adjacent to the cytotrophoblast are called amnioblasts; together with the rest of the epiblast, they line the amniotic cavity
(Figs. 3.1 and 3.3). The endometrial stroma adjacent to the implantation site
is edematous and highly vascular. The large, tortuous glands secrete abundant
glycogen and mucus.
Chapter 3: Second Week of Development: Bilaminar Germ Disc
53
Figure 3.3 A 9-day human blastocyst. The syncytiotrophoblast shows a large number
of lacunae. Flat cells form the exocoelomic membrane. The bilaminar disc consists of
a layer of columnar epiblast cells and a layer of cuboidal hypoblast cells. The original
surface defect is closed by a ﬁbrin coagulum.
Day 9
The blastocyst is more deeply embedded in the endometrium, and the penetration defect in the surface epithelium is closed by a ﬁbrin coagulum (Fig. 3.3).
The trophoblast shows considerable progress in development, particularly at
the embryonic pole, where vacuoles appear in the syncytium. When these vacuoles fuse, they form large lacunae, and this phase of trophoblast development
is thus known as the lacunar stage (Fig. 3.3).
At the abembryonic pole, meanwhile, ﬂattened cells probably originating
from the hypoblast form a thin membrane, the exocoelomic (Heuser’s) membrane, that lines the inner surface of the cytotrophoblast (Fig. 3.3). This membrane, together with the hypoblast, forms the lining of the exocoelomic cavity,
or primitive yolk sac.
Days 11 and 12
By the 11th to 12th day of development, the blastocyst is completely embedded
in the endometrial stroma, and the surface epithelium almost entirely covers
54
Part One: General Embryology
Figure 3.4 Human blastocyst of approximately 12 days. The trophoblastic lacunae at
the embryonic pole are in open connection with maternal sinusoids in the endometrial
stroma. Extraembryonic mesoderm proliferates and ﬁlls the space between the exocoelomic membrane and the inner aspect of the trophoblast.
the original defect in the uterine wall (Figs. 3.4 and 3.5). The blastocyst now
produces a slight protrusion into the lumen of the uterus. The trophoblast is
characterized by lacunar spaces in the syncytium that form an intercommunicating network. This network is particularly evident at the embryonic pole; at
the abembryonic pole, the trophoblast still consists mainly of cytotrophoblastic
cells (Figs. 3.4 and 3.5).
Concurrently, cells of the syncytiotrophoblast penetrate deeper into the
stroma and erode the endothelial lining of the maternal capillaries. These capillaries, which are congested and dilated, are known as sinusoids. The syncytial
lacunae become continuous with the sinusoids and maternal blood enters the
lacunar system (Fig. 3.4). As the trophoblast continues to erode more and more
sinusoids, maternal blood begins to ﬂow through the trophoblastic system, establishing the uteroplacental circulation.
In the meantime, a new population of cells appears between the inner surface of the cytotrophoblast and the outer surface of the exocoelomic
Chapter 3: Second Week of Development: Bilaminar Germ Disc
55
Figure 3.5 Fully implanted 12-day human blastocyst (×100). Note maternal blood cells
in the lacunae, the exocoelomic membrane lining the primitive yolk sac, and the hypoblast and epiblast.
cavity. These cells, derived from yolk sac cells, form a ﬁne, loose connective tissue, the extraembryonic mesoderm, which eventually ﬁlls all of the
space between the trophoblast externally and the amnion and exocoelomic
membrane internally (Figs. 3.4 and 3.5). Soon, large cavities develop in the
extraembryonic mesoderm, and when these become conﬂuent, they form
a new space known as the extraembryonic coelom, or chorionic cavity
(Fig. 3.4). This space surrounds the primitive yolk sac and amniotic cavity except where the germ disc is connected to the trophoblast by the connecting stalk
(Fig. 3.6). The extraembryonic mesoderm lining the cytotrophoblast and amnion is called the extraembryonic somatopleuric mesoderm; the lining covering the yolk sac is known as the extraembryonic splanchnopleuric mesoderm
(Fig. 3.4).
Growth of the bilaminar disc is relatively slow compared with that of the trophoblast; consequently, the disc remains very small (0.1–0.2 mm). Cells of the
endometrium, meanwhile, become polyhedral and loaded with glycogen and
lipids; intercellular spaces are ﬁlled with extravasate, and the tissue is edematous. These changes, known as the decidua reaction, at ﬁrst are conﬁned to the
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Part One: General Embryology
Figure 3.6 A 13-day human blastocyst. Trophoblastic lacunae are present at the embryonic as well as the abembryonic pole, and the uteroplacental circulation has begun. Note
the primary villi and the extraembryonic coelom or chorionic cavity. The secondary
yolk sac is entirely lined with endoderm.
area immediately surrounding the implantation site but soon occur throughout
the endometrium.
Day 13
By the 13th day of development, the surface defect in the endometrium has
usually healed. Occasionally, however, bleeding occurs at the implantation site
as a result of increased blood ﬂow into the lacunar spaces. Because this bleeding
occurs near the 28th day of the menstrual cycle, it may be confused with
Chapter 3: Second Week of Development: Bilaminar Germ Disc
57
Figure 3.7 Section through the implantation site of a 13-day embryo. Note the amniotic
cavity, yolk sac, and exocoelomic cyst in the chorionic cavity. Most of the lacunae are
ﬁlled with blood.
normal menstrual bleeding and, therefore, cause inaccuracy in determining
the expected delivery date.
The trophoblast is characterized by villous structures. Cells of the cytotrophoblast proliferate locally and penetrate into the syncytiotrophoblast,
forming cellular columns surrounded by syncytium. Cellular columns with
the syncytial covering are known as primary villi (Figs. 3.6 and 3.7) (see
Chapter 4).
In the meantime, the hypoblast produces additional cells that migrate along
the inside of the exocoelomic membrane (Fig. 3.4). These cells proliferate and
gradually form a new cavity within the exocoelomic cavity. This new cavity is
known as the secondary yolk sac or deﬁnitive yolk sac (Figs. 3.6 and 3.7). This
yolk sac is much smaller than the original exocoelomic cavity, or primitive yolk
sac. During its formation, large portions of the exocoelomic cavity are pinched
off. These portions are represented by exocoelomic cysts, which are often
found in the extraembryonic coelom or chorionic cavity (Figs. 3.6 and 3.7).
Meanwhile, the extraembryonic coelom expands and forms a large cavity,
the chorionic cavity. The extraembryonic mesoderm lining the inside of the
cytotrophoblast is then known as the chorionic plate. The only place where
extraembryonic mesoderm traverses the chorionic cavity is in the connecting
stalk (Fig. 3.6). With development of blood vessels, the stalk becomes the
umbilical cord.
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Part One: General Embryology
CLINICAL CORRELATES
Abnormal Implantation
The syncytiotrophoblast is responsible for hormone production (see Chapter 6), including human chorionic gonadotropin (hCG). By the end of the
second week, quantities of this hormone are sufﬁcient to be detected by radioimmunoassays, which serve as the basis for pregnancy testing.
Because 50% of the implanting embryo’s genome is derived from the
father, it is a foreign body that potentially should be rejected by the maternal system. Recent evidence suggests that a combination of factors protects
the conceptus, including production of immunosuppressive cytokines and proteins and the expression of an unusual major histocompatibility complex class
IB molecule (HLA-G) that blocks recognition of the conceptus as foreign tissue.
If the mother has autoimmune disease, for example systemic lupus erythematosus, antibodies generated by the disease may attack the conceptus and
reject it.
Abnormal implantation sites sometimes occur even within the uterus.
Normally the human blastocyst implants along the anterior or posterior wall of
the body of the uterus. Occasionally the blastocyst implants close to the internal opening os (opening) (Fig. 3.8) of the cervix, so that later in development,
Intestinal loop
Mesentery
Body of
uterus
1
3
Uterine tube
4
2
5
Ampulla
6
Internal os
of uterus
Fimbriae
Figure 3.8 Abnormal implantation sites of the blastocyst. 1, implantation in the abdominal cavity. The ovum most frequently implants in the rectouterine cavity (Douglas’
pouch) but may implant at any place covered by peritoneum. 2, implantation in the ampullary region of the tube. 3, tubal implantation. 4, interstitial implantation, that is, in
the narrow portion of the uterine tube. 5, implantation in the region of the internal os,
frequently resulting in placenta previa. 6, ovarian implantation.
Chapter 3: Second Week of Development: Bilaminar Germ Disc
59
Figure 3.9 Tubal pregnancy. Embryo is approximately 2 months old and is about to
escape through a rupture in the tubal wall.
the placenta bridges the opening (placenta previa) and causes severe, even
life-threatening bleeding in the second part of pregnancy and during delivery.
Occasionally, implantation takes place outside the uterus, resulting in extrauterine pregnancy, or ectopic pregnancy. Ectopic pregnancies may occur
at any place in the abdominal cavity, ovary, or uterine tube (Fig. 3.8). However,
95% of ectopic pregnancies occur in the uterine tube, and most of these are in
the ampulla (Fig. 3.9). In the abdominal cavity, the blastocyst most frequently
attaches itself to the peritoneal lining of the rectouterine cavity, or Douglas’
pouch (Fig. 3.10). The blastocyst may also attach itself to the peritoneal covering of the intestinal tract or to the omentum. Sometimes the blastocyst
develops in the ovary proper, causing a primary ovarian pregnancy. In most
ectopic pregnancies, the embryo dies about the second month of gestation,
causing severe hemorrhaging and abdominal pain in the mother.
Abnormal blastocysts are common. For example, in a series of 26 implanted blastocysts varying in age from 7.5 to 17 days recovered from patients
of normal fertility, nine (34.6%) were abnormal. Some consisted of syncytium
only; others showed varying degrees of trophoblastic hypoplasia. In two, the
embryoblast was absent, and in some, the germ disc showed an abnormal
orientation.
It is likely that most abnormal blastocysts would not have produced any
sign of pregnancy because their trophoblast was so inferior that the corpus
luteum could not have persisted. These embryos probably would have been
60
Part One: General Embryology
Figure 3.10 Midline section of bladder, uterus, and rectum to show an abdominal pregnancy in the rectouterine (Douglas’) pouch.
aborted with the next menstrual ﬂow, and therefore, pregnancy would not
have been detected. In some cases, however, the trophoblast develops and
forms placental membranes, although little or no embryonic tissue is present.
Such a condition is known as a hydatidiform mole. Moles secrete high levels of
hCG and may produce benign or malignant (invasive mole, choriocarcinoma)
tumors.
Genetic analysis of hydatidiform moles indicates that although male and
female pronuclei may be genetically equivalent, they may be different functionally. This evidence is derived from the fact that while cells of moles are
diploid, their entire genome is paternal. Thus, most moles arise from fertilization of an oocyte lacking a nucleus followed by duplication of the male
chromosomes to restore the diploid number. These results also suggest that
paternal genes regulate most of the development of the trophoblast, since in
moles, this tissue differentiates even in the absence of a female pronucleus.
Other examples of functional differences in maternal and paternal genes
are provided by the observation that certain genetic diseases depend on
whether the defective or missing gene is inherited from the father or the
mother. For example, inheritance of a deletion on chromosome 15 from a father produces Prader-Willi syndrome, whereas inheritance of the same defect
from the mother results in Angelman syndrome. This phenomenon, in which
Chapter 3: Second Week of Development: Bilaminar Germ Disc
61
there is differential modiﬁcation and/or expression of homologous alleles or
chromosome regions, depending on the parent from whom the genetic material is derived, is known as genomic imprinting. Imprinting involves autosomes and sex chromosomes (in all female mammals, one X chromosome
is inactivated in somatic cells and forms a chromatin-positive body [Barr
body]) and is modulated by deoxyribonucleic acid (DNA) methylation. Certain diseases, such as Huntington’s chorea, neuroﬁbromatosis, familial cancer
disorders (Wilms’ tumors, familial retinoblastoma), and myotonic dystrophy,
also involve imprinting. Fragile X syndrome, the leading cause of inherited
mental retardation, may be another example of a condition based on imprinting (see Chapter 1).
Preimplantation and postimplantation reproductive failure occurs oft